Expression and characterization of HIV-1 envelope protein associated with a broadly reactive neutralizing antibody response

ABSTRACT

The present invention relates to HIV-1 envelope proteins from a donor with non-progressive HIV-1 infection whose serum contains broadly cross-reactive, primary virus neutralizing antibody. The invention also relates to isolated or purified proteins and protein fragments that share certain amino acids at particular positions with the foregoing HIV-1 proteins.

RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 09/762,261 (filed Feb. 5, 2001), which is a U.S. National PhaseApplication of International Application PCT/US99/17596 (filed on Aug.4, 1999), which claims the benefit of U.S. Provisional Application60/095,267 (filed Aug. 4, 1998), all of which are herein incorporated byreference in their entirety.

ACKNOWLEDGMENT OF FEDERAL SUPPORT

The present invention arose in part from research funded by thefollowing federal grant monies: NIH A137436 and A144339, and USUHSR087E2

TECHNICAL FIELD

The present invention relates to HIV-1 envelope proteins and peptidesderived from the donor of the Neutralizing Reference Human Serum (2)which is noted for its capacity to neutralize primary HIV isolates ofvaried subtypes.

BACKGROUND OF THE INVENTION

The development of a successful vaccine against HIV infection or avaccine agent capable of preventing HIV disease progression has been apublic health goal for over 15 years. One of the immune responses thatmay be required to elicit a protective immune response against HIVinfection is the generation of antibodies that are virus neutralizing.

The target of HIV-1 neutralizing antibodies (NA) is the envelopeglycoprotein complex. This complex is a multimeric structure composed ofthree or four copies each of the gp120 surface and gp41 transmembraneglycoproteins (Luciw, 1996). There are a number of neutralizationdomains on each of the three or four heterodimeric components of thecomplex (Thali et al., 1992, 1993; Zwart et al., 1991; Moore et al.,1993; Trkola et al., 1996; Muster et al., 1993; Cotropia et al., 1996;Sabri et al., 1996). The amino acid compositions of the proteins varysubstantially from strain to strain. Some of the neutralization domainsare in regions which tend to vary greatly, while others are in regionswhich tend to be highly conserved. The variable neutralization domainsinclude those in variable (V) regions 1, 2, and 3 of gp120, while theconserved domains include the primary receptor binding site, and otherepitopes in gp120 and gp41. Amino acid sequence variation is undoubtedlythe explanation for the variation that is seen in specificity ofneutralization sensitivity among virus strains. However, it has not beenpossible to classify antigenic subtypes of HIV-1 based on geneticanalyses, and various regions of the envelope complex even outside ofthe neutralization domains have been shown to contribute to antigenicvariability (Thali et al., 1994; Back et al., 1993).

Recent findings indicate that the neutralization of primary isolates ofHIV may be mediated primarily by antibodies directed against non-V3region epitopes expressed on the oligomeric complex but not on monomericgp120, while laboratory adapted strains are more readily neutralized byantibodies directed against V3 (Hioe et al., 1997; VanCott et al.,1997). The identity of the non-V3 epitopes recognized on primaryisolates is not established. The presence of antibodies which havebroadly neutralizing activity against primary isolates of many subtypesof HIV-1 in sera from infected people is unusual, but the nature of theenvelope proteins in individuals with such antibodies may be of interestfor defining the epitopes which may be broadly immunogenic in vaccines.

SUMMARY OF THE INVENTION

The present inventors have cloned and characterized the envelope genesfrom the donor of human serum which is noted for its capacity toneutralize primary HIV isolates of various subtype (Vujcic, et al. 1995,D'Souza et al., 1991).

The invention includes an isolated HIV envelope protein or fragmentthereof which, when injected into a mammal, induces the production ofbroadly cross-reactive neutralizing anti-serum against multiple strainsof HIV-1.

The invention further includes an isolated HIV envelope protein orfragment thereof comprising a proline at a position corresponding toamino acid residue 313, a methionine at a position corresponding toamino acid residue 314 and a glutamine at a position corresponding toamino acid residue 325 of SEQ ID NO:1.

In another embodiment, the invention includes an isolated HIV envelopeglycoprotein or fragment thereof comprising an alanine at a positioncorresponding to amino acid residue 167 of SEQ ID NO:1.

The invention also includes an isolated HIV envelope protein comprisingthe amino acid sequence of SEQ ID NO:1 as well as an isolated nucleicacid molecule encoding the envelope protein, which was deposited withthe American Type Culture Collection (ATCC) under accession No.PTA-7237.

Compositions for eliciting an immune response, such as vaccines,immunogenic compositions and attenuated viral vaccine delivery vectorscomprising the envelope proteins, peptides and nucleic acids encodingsuch proteins and peptides of the invention are also included. Methodsfor generating antibodies in a mammal comprising administering one ormore of these proteins, peptides and nucleic acids, in an amountsufficient to induce the production of the antibodies, is also includedin the invention.

The invention also comprises a diagnostic reagent comprising one or moreof the isolated HIV-1 envelope proteins and methods for detectingbroadly cross-reactive neutralizing anti-serum against multiple strainsof HIV-1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Phylogenetic analysis of the gp120 and gp41 nucleotide codingsequences of clone R2. Alignments were performed using the Clustialalgorithm of Higgins and Sharp in the program DNA Star (Higgins et al.,1989; Saitou et al., 1987; Myers et al., 1988). The graphs at the bottomof the two figures indicate the percent similarity distances representedby the dendograms. Gene bank accession numbers for the sequencesrepresented are: MW 959, U08453; MW960, U08454; D747, X65638; BR020,U27401; BR029, U27413; RU131, U30312; UG975, U27426; AD8, M60472; HXB,K03455; NDK, M27323; Z2Z6, M22639; UG021, U27399; CM235, L03698; TH022,U09139; TH006, U08810; UG275, L22951; SF1703, M66533; RW020, U08794;RW009, U08793; U455, M62320; and Z321, M15896.

FIG. 2: Neutralization of clade B viruses and pseudoviruses by sera from10 male residents of the Baltimore/Washington, D.C. area collected from1985-1990 in the Multicenter AIDS Cohort Study. The P9 and P10 viruses(P9-V and P10-V) are primary isolates from two of the serum donors(Quinnan et al., 1998). The neutralization assays were performed in PM1cells, as described in the Examples. Each point represents the resultsobtained with an individual serum. The open bars represent the standarddeviations about the geometric means, indicated by the midlines. Thenumbers above the results obtained using pseudoviruses indicate theprobabilities obtained from testing the null hypothesis by paired ttesting comparing the individual pseudoviruses to R2.

FIG. 3 (A): Inhibition of Reference 2-mediated neutralization ofpseudoviruses by synthetic V3 peptides. The neutralization endpoints for90% neutralization were calculated as described previously (Quinnan etal., 1999; Quinnan et al., 1998; Zhang et al., 1999; Park et al., 1998).Results shown are means of triplicate determinations. Dose-responseeffects of R2 linear 17-mer (open square) and cyclic (closed square)(SEQ ID NO:2) and the 93TH966.8 cyclic (shaded square) (SEQ ID NO:3) V3peptides on neutralization of clone R2 pseudovirus. The peptideconcentrations are 3×10 raised to the indicated power.

FIG. 3 (B): Comparative inhibitory effects of peptides on neutralizationof R2 and MN (clone V5) pseudoviruses. All peptides were tested at 15μg/ml. The linear peptides (L) corresponded to the apical sequences ofthe respective V3 loops. The cyclic peptides (C) corresponded to thefull lengths of the respective V3 regions of the different strains.Neutralization in the absence of peptide (None), is also shown.

FIG. 4 (A): Effect of cyclic R2 V3 peptide on neutralization ofpseudoviruses. Fold inhibition of neutralization was calculated as theratio of the 50% neutralization titer obtained in the absence of peptidecompared to that obtained in the presence of cyclic R2 V3 peptide (15μg/ml). All assays were performed in triplicate. Neutralization titerswere calculated at the midpoints of the infectivity inhibition curves,since the curves tended to be most parallel in this region. Similarresults were obtained comparing 90% neutralization endpoints. Peptideinhibition of neutralization of R2 pseudovirus by sera from MACS donors(donor numbers 1-10), two assays each, and by Reference 2. Results areshown for two determinations for each serum from the MACS donors and for12 assays of Reference 2 performed during the same time intervals as theother experiments shown in panels (A) and (B).

FIG. 4 (B): Peptide inhibition of neutralization of pseudovirusesexpressing MACS patient envelopes (patient numbers 3, 4, 6, 8, 9, and10) by Reference 2. Results of two or three separate assays of eachpseudovirus are shown.

MODES OF CARRYING OUT THE INVENTION

General Description

A goal of immunization against HIV is to induce neutralizing antibody(NA) responses broadly reactive against diverse strains of virus. Thepresent inventors have studied envelope protein from a donor withnon-progressive HIV-1 infection whose serum contains broadlycross-reactive, primary virus NA. DNA was extracted from lymphocytes,which had been collected approximately six and twelve months prior tothe time of collection of the cross reactive serum, env genes weresynthesized by nested PCR, cloned, expressed on pseudoviruses, andphenotyped in NA assays. Two clones from each time point had identicalV3 region nucleotide sequences, utilized CCR5 but not CXCR4 for cellentry, and had similar reactivities with two reference sera. Analysis ofthe full nucleotide sequence of one clone demonstrated it to be subtypeB, with a predicted GPGRAF apical V3 sequence, normal predictedglycosylation, and an intact reading frame. Infectivity assays of R2pseudovirus in HOS cells expressing CD4 and various coreceptorsdemonstrated the envelope to be CCR5 dependent. R2 pseudovirus wascompared to others expressing env genes of various clades forneutralization by sera from donors in the United States (presumed orknown subtype B infections), and from individuals infected with subtypesA, C, and E viruses. Neutralization by the sera from donors in theUnited States of pseudoviruses expressing R2 and other clade B envs wassimilarly low to moderate, although R2 was uniquely neutralized by all.R2 was neutralized by sera from people infected with clades A-F, whileother clade B, D, E and G pseudoviruses were neutralized less often. Onehighly sensitive clade C pseudovirus was neutralized by all the sera,although the titers varied more than 250-fold. The results suggest thatthe epitope(s) which induced the cross-clade reactive NA in Donor 2 maybe expressed on the R2 envelope.

The present invention relates to HIV-1 envelope proteins from this donorwho had non-progressive HIV-1 infection whose serum contains broadlycross-reactive, primary virus neutralizing antibody. The invention alsorelates to isolated or purified proteins and protein fragments thatshare certain amino acids at particular positions with the foregoingHIV-1 proteins.

Specific Embodiments

Proteins and Peptides

Proteins and peptides of the invention include the full length envelopeprotein having the amino acid sequence of Table 3 (SEQ ID NO:1), gp120having the amino acid sequence corresponding to gp120 in Table 3 (aminoacids: 1-520 of SEQ ID NO:1), gp41 having the amino acid sequencecorresponding to gp41 in Table 3 (amino acids 521-866 of SEQ ID NO:1),as well as polypeptides and peptides corresponding to the V3 domain andother domains such as V1/V2, C3, V4, C4 and V5. These domains correspondto the following amino acid residues of SEQ ID NO:1: DOMAIN AMINO ACIDRESIDUES C1  30-124 V1 125-162 V2 163-201 C2 202-300 V3 301-336 C3337-387 V4 388-424 C4 425-465 V5 466-509 C5 510-520

Polypeptides and peptides comprising any single domain may be ofvariable length but include the amino acid residues of Table 3 (SEQ IDNO:1) which differ from previously sequenced envelope proteins. Forinstance, peptides of the invention which include all or part of the V3domain may comprise the sequence: PM X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀Q(SEQ ID NO:5), wherein X₁-X₁₀ are any natural or non-natural amino acids(P refers to Proline, M refers to methionine and Q refers to Glutamine).Non-natural amino acids include, for example, beta-alanine (beta-Ala),or other omega-amino acids, such as 3-amino propionic, 2,3-diaminopropionic (2,3-diaP), 4-amino butyric and so forth, alpha-aminisobutyricacid (Aib), sarcosine (Sat), ornithine (Orn), citrulline (Cit),t-butylalanine (t-BuA), t-butylglycine (t-BuG), N-methylisoleucine(N-Melle), phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine(Nle), cysteic acid (Cya) 2-naphthylalanine (2-Nal);1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);beta-2-thienylalanine (Thi); and methionine sulfoxide (MSO). Preferably,peptides of the invention are 60%, 70%, 80% or more preferably, 90%identical to the V3 region of the HIV envelope protein of Table 3 (SEQID NO:1). Accordingly, V3 peptides of the invention comprise about 13amino acids but may be 14, 15, 17, 20, 25, 30, 35, 36, 39, 40, 45, 50 ormore amino acids in length. In one embodiment, a V3 peptide of 13 aminoacids in length consists of the sequence PMGPGRAFYTTGQ (amino acids313-325 of Table 3 (SEQ ID NO:1).

In another embodiment of the invention, polypeptides and peptidescomprising all or part of the V1/V2 domain comprise an amino acidsequence with an alanine residue at a position corresponding to aminoacid 167 Table 3 (SEQ ID NO:1). For instance, peptides of the inventionspanning the V1/V2 domain may comprise the sequence FNIATSIG (residues164-171 of SEQ ID NO:1) and may be about 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50 or more amino acids in length. As used herein, “at a positioncorresponding to” refers to amino acid positions in HIV envelopeproteins or peptides of the invention which are equivalent to a givenamino acid residue in the sequence of Table 1 (SEQ ID NO:1) in thecontext of the surrounding residues.

The peptides of the present invention may be prepared by any knowntechniques. Conveniently, the peptides may be prepared using thesolid-phase synthetic technique initially described by Merrifield(1965), which is incorporated herein by reference. Other peptidesynthesis techniques may be found, for example, in Bodanszky et al.,Peptide Synthesis, 2d ed. (New York, Wiley, 1976).

Nucleic acids and Recombinant Expression of Peptide or Proteins

Proteins and peptides of the invention may be prepared by any availablemeans, including recombinant expression of the desired protein orpeptide in eukaryotic or prokaryotic host cells (see U.S. Pat. No.5,696,238). Methods for producing proteins or peptides of the inventionfor purification may employ conventional molecular biology,microbiology, and recombinant DNA techniques within the ordinary skilllevel of the art. Such techniques are explained fully in the literature.See, for example, Maniatis et al., Molecular Cloning: A LaboratoryManual, 2d ed. (Cold Spring Harbor, Cold Spring Harbor Laboratory Press,1989); Glover, DNA Cloning: A Practical Approach, Vols. 1-4 (Oxford, IRLPress, 1985); Gait, Oligonucleotide Synthesis: A Practical Approach(Oxford, IRL Press, 1984); Hames & Higgins, Nucleic Acid Hybridisation:A Practical Approach (Oxford, IRL Press, 1985); Freshney, Animal CellCulture. A Practical Approach (Oxford, IRL Press, 1992); Perbal, APractical Guide To Molecular Cloning (New York, Wiley, 1984).

The present invention further provides nucleic acid molecules thatencode the proteins or peptides of the invention. Such nucleic acidmolecules can be in an isolated form, or can be operably linked toexpression control elements or vector sequences. The present inventionfurther provides host cells that contain the vectors via transformation,transfection, electroporation or any other art recognized means ofintroducing a nucleic acid into a cell.

As used herein, a “cell line” is a clone of a primary cell that iscapable of stable growth in vitro for many generations.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

As used herein, “naked DNA” means nucleic acid molecules that are freefrom viral particles, particularly retroviral particles. This term alsomeans nucleic acid molecules which are free from facilitator agentsincluding but not limited to the group comprising: lipids, liposomes,extracellular matrix-active enzymes, saponins, lectins, estrogeniccompounds and steroidal hormones, hydroxylated lower alkyls, dimethylsulfoxide (DMSO) and urea.

As used herein, a “nucleic acid molecule” refers to the polymeric formof deoxyribonucleotides (adenine, guanine, thymine, and/or cytosine) ineither its single stranded form, or in double-stranded helix as well asRNA. This term refers only to the primary and secondary structure of themolecule and is not limited to any particular tertiary form. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (e.g., the strand having a sequencehomologous to the mRNA). Transcriptional and translational controlsequences are DNA regulatory sequences, such as promoters, enhancers,polyadenylation signals, terminators, and the like, that provide for theexpression of a coding sequence in a host cell.

As used herein, a “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is bounded (inclusively) at its3′ terminus by the transcription initiation site and extends upstream(5′ direction) to include the minimum number of bases or elementsnecessary to initiate transcription at levels detectable abovebackground. Within the promoter sequence will be found a transcriptioninitiation site, as well as protein binding domains responsible for thebinding of RNA polymerase. Eukaryotic promoters will often, but notalways, contain “TATA” boxes and “CAT” boxes.

As used herein, a “replicon” is any genetic element (e.g., plasmid,chromosome, virus) that functions as an autonomous unit of DNAreplication in vivo; i.e., capable of replication under its own control.

A “signal sequence” can be included before the coding sequence or thenative 29 amino acid signal sequence from the envelope protein of Table3 may be used. This sequence encodes a signal peptide, N-terminal to thepolypeptide, that communicates to the host cell to direct thepolypeptide to the cell surface or secrete the polypeptide into themedia. This signal peptide is clipped off by the host cell before theprotein leaves the cell. Signal sequences can be found associated with avariety of proteins native to prokaryotes and eukaryotes. For instance,alpha-factor, a native yeast protein, is secreted from yeast, and itssignal sequence can be attached to heterologous proteins to be secretedinto the media (See U.S. Pat. No. 4,546,082, and EP 0116201). Further,the alpha-factor and its analogs have been found to secrete heterologousproteins from a variety of yeast, such as Saccharomyces andKluyveromyces, (EP 88312306.9; EP 0324274 publication, and EP 0301669).An example for use in mammalian cells is the tPA signal used forexpressing Factor VIIIc light chain.

As used herein, DNA sequences are “substantially homologous” when atleast about 85% (preferably at least about 90% and most preferably atleast about 95%) of the nucleotides match over the defined length of theDNA sequences. Sequences that are substantially homologous can beidentified in a Southern hybridization experiment under, for example,stringent conditions as defined for that particular system. Definingappropriate hybridization conditions is within the skill of the art.See, for example, Maniatis et al., supra.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA as been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, for example, the transforming DNAmay be maintained on an episomal element such as a plasmid or viralvector. With respect to eukaryotic cells, a stably transformed cell isone in which the transforming DNA has become integrated into achromosome so that it is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the transforming DNA.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

As used herein, a “vector” is a replicon, such as plasmid, phage orcosmid, to which another DNA segment may be attached so as to bringabout the replication of the attached segment.

Vectors are used to simplify manipulation of the DNA which encodes theHIV proteins or peptides, either for preparation of large quantities ofDNA for further processing (cloning vectors) or for expression of theHIV proteins of peptides (expression vectors). Vectors compriseplasmids, viruses (including phage), and integrated DNA fragments, i.e.,fragments that are integrated into the host genome by recombination.Cloning vectors need not contain expression control sequences. However,control sequences in an expression vector include transcriptional andtranslational control sequences such as a transcriptional promoter, asequence encoding suitable ribosome binding sites, and sequences whichcontrol termination of transcription and translation. The expressionvector should preferably include a selection gene to facilitate thestable expression of HIV gene and/or to identify transformants. However,the selection gene for maintaining expression can be supplied by aseparate vector in cotransformation systems using eukaryotic host cells.

Suitable vectors generally will contain replicon (origins ofreplication, for use in non-integrative vectors) and control sequenceswhich are derived from species compatible with the intended expressionhost. By the term “replicable” vector as used herein, it is intended toencompass vectors containing such replicons as well as vectors which arereplicated by integration into the host genome. Transformed host cellsare cells which have been transformed or transfected with vectorscontaining HIV peptide or protein encoding DNA. The expressed HIVproteins or peptides may be secreted into the culture supernatant, underthe control of suitable processing signals in the expressed peptide,e.g. homologous or heterologous signal sequences.

Expression vectors for host cells ordinarily include an origin ofreplication, a promoter located upstream from the HIV protein or peptidecoding sequence, together with a ribosome binding site, apolyadenylation site, and a transcriptional termination sequence. Thoseof ordinary skill will appreciate that certain of these sequences arenot required for expression in certain hosts. An expression vector foruse with microbes need only contain an origin of replication recognizedby the host, a promoter which will function in the host, and a selectiongene.

Commonly used promoters are derived from polyoma, bovine papillomavirus, CMV (cytomegalovirus, either murine or human), Rouse sarcomavirus, adenovirus, and simian virus 40 (SV40). Other control sequences(e.g., terminator, polyA, enhancer, or amplification sequences) can alsobe used.

An expression vector is constructed so that the HIV protein or peptidecoding sequence is located in the vector with the appropriate regulatorysequences, the positioning and orientation of the coding sequence withrespect to the control sequences being such that the coding sequence istranscribed and translated under the “control” of the control sequences(i.e., RNA polymerase which binds to the DNA molecule at the controlsequences transcribes the coding sequence). The control sequences may beligated to the coding sequence prior to insertion into a vector, such asthe cloning vectors described above. Alternatively, the coding sequencecan be cloned directly into an expression vector which already containsthe control sequences and an appropriate restriction site. If theselected host cell is a mammalian cell, the control sequences can beheterologous or homologous to the HIV coding sequence, and the codingsequence can either be genomic DNA containing introns or cDNA.

Higher eukaryotic cell cultures may be used to express the proteins ofthe present invention, whether from vertebrate or invertebrate cells,including insects, and the procedures of propagation thereof are known.See, for example, Kruse & Patterson, Tissue Culture (New York, AcademicPress, 1973).

Suitable host cells for expressing HIV proteins or peptides in highereukaryotes include: monkey kidney CVI line transformed by SV40 (COS-7,ATCC CRL1651); baby hamster kidney cells (BHK, ATCC CRL10); Chinesehamster ovary-cells-DHFR (Urlaub & Chasin, 1980); mouse Sertoli cells(Mather, 1980); monkey kidney cells (CVI ATCC CCL70); African greenmonkey kidney cells (VER076, ATCC CRL1587); human cervical carcinomacells (HeLa, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL34); buffalorat liver cells (BRL3A, ATCC CRL1442); human lung cells (W138, ATCCCCL75); human liver cells (HepG2, HB8065); mouse mammary tumor (MMT060652, ATCC CCL51); rat hepatoma cells (Baumann et al., 1980) and TR1cells (Mather et al., 1982).

It will be appreciated that when expressed in mammalian tissue, therecombinant HIV gene products may have higher molecular weights thanexpected due to glycosylation. It is therefore intended that partiallyor completely glycosylated forms of HIV preproteins or peptides havingmolecular weights somewhat different from 160, 120 or 41 kD are withinthe scope of this invention.

Other preferred expression vectors are those for use in eukaryoticsystems. An exemplary eukaryotic expression system is that employingvaccinia virus, which is well-known in the art. See, for example, Macketet al. (1984); Glover, supra; and WO 86/07593. Yeast expression vectorsare known in the art. See, for example, U.S. Pat. Nos. 4,446,235;4,443,539; 4,430,428; and EP 103409; EP 100561; EP 96491.

Another preferred expression system is vector pHSI, which transformsChinese hamster ovary cells (see WO 87/02062). Mammalian tissue may becotransformed with DNA encoding a selectable marker such asdihydrofolate reductase (DHFR) or thymidine kinase and DNA encoding theHIV protein or peptide. If wild type DHFR gene is employed, it ispreferable to select a host cell which is deficient in DHFR, thuspermitting the use of the DHFR coding sequence as marker for successfultransfection in hgt medium, which lacks hypoxanthine, glycine, andthymidine. An appropriate host cell in this case is the Chinese hamsterovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub & Chasin, (1980).

Depending on the expression system and host selected, HIV proteins orpeptides are produced by growing host cells transformed by an exogenousor heterologous DNA construct, such as an expression vector describedabove under conditions whereby the HIV protein is expressed. The HIVprotein or peptide is then isolated from the host cells and purified. Ifthe expression system secretes the protein or peptide into the growthmedia, the protein can be purified directly from cell-free media. Theselection of the appropriate growth conditions and initial cruderecovery methods are within the skill of the art.

Once a coding sequence for an HIV protein or peptide of the inventionhas been prepared or isolated, it can be cloned into any suitable vectorand thereby maintained in a composition of cells which is substantiallyfree of cells that do not contain an HIV coding sequence. Numerouscloning vectors are known to those of skill in the art. Examples ofrecombinant DNA vectors for cloning and host cells which they cantransform include the various bacteriophage lambda vectors (E. coli),pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria),pGV1106 (gram-negative bacteria), pLAFR1 (gram-negative bacteria),pME290 (non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), plJ61 (Streptomyces). pUC6 (Streptomyces),actinophage, fC31 (Streptomyces). YIpS (Saccharomyces), YCp19(Saccharomyces), and bovine papilloma virus (mammalian cells). Seegenerally, Glover, supra; T. Maniatis et al., supra; and Perbal, supra.

Fusion Proteins

HIV envelope fusion proteins and methods for making such proteins havebeen previously described (U.S. Pat. No. 5,885,580). It is now arelatively straight forward technology to prepare cells expressing aforeign gene. Such cells act as hosts and may include, for the fusionproteins of the present invention, yeasts, fungi, insect cells, plantscells or animals cells. Expression vectors for many of these host cellshave been isolated and characterized, and are used as starting materialsin the construction, through conventional recombinant DNA techniques, ofvectors having a foreign DNA insert of interest. Any DNA is foreign ifit does not naturally derive from the host cells used to express the DNAinsert. The foreign DNA insert may be expressed on extrachromosomalplasmids or after integration in whole or in part in the host cellchromosome(s), or may actually exist in the host cell as a combinationof more than one molecular form. The choice of host cell and expressionvector for the expression of a desired foreign DNA largely depends onavailability of the host cell and how fastidious it is, whether the hostcell will support the replication of the expression vector, and otherfactors readily appreciated by those of ordinary skill in the art.

The foreign DNA insert of interest comprises any DNA sequence coding forfusion proteins including any synthetic sequence with this codingcapacity or any such cloned sequence or combination thereof. Forexample, fusion proteins coded and expressed by an entirely recombinantDNA sequence is encompassed by this invention but not to the exclusionof fusion proteins peptides obtained by other techniques.

Vectors useful for constructing eukaryotic expression systems for theproduction of fusion proteins comprise the fusion protein's DNAsequence, operatively linked thereto with appropriate transcriptionalactivation DNA sequences, such as a promoter and/or operator. Othertypical features may include appropriate ribosome binding sites,termination codons, enhancers, terminators, or replicon elements. Theseadditional features can be inserted into the vector at the appropriatesite or sites by conventional splicing techniques such as restrictionendonuclease digestion and ligation.

Yeast expression systems, which are the preferred variety of recombinanteukaryotic expression system, generally employ Saccharomyces cerevisiaeas the species of choice for expressing recombinant proteins. Otherspecies of the genus Saccharomyces are suitable for recombinant yeastexpression system, and include but are not limited to carlsbergensis,uvarum, rouxii, montanus, kluyveri, elongisporus, norbensis, oviformis,and diastaticus. Saccharomyces cerevisiae and similar yeasts possesswell known promoters useful in the construction of expression systemsactive in yeast, including but not limited to GAP, GAL10, ADH2, PHO5,and alpha mating factor.

Yeast vectors useful for constructing recombinant yeast expressionsystems for expressing fusion proteins include, but are not limited to,shuttle vectors, cosmid plasmids, chimeric plasmids, and those havingsequences derived from two micron circle plasmids. Insertion of theappropriate DNA sequence coding for fusion proteins into these vectorswill, in principle, result in a useful recombinant yeast expressionsystem for fusion proteins where the modified vector is inserted intothe appropriate host cell, by transformation or other means. Recombinantmammalian expression system are another means of producing the fusionproteins for the vaccines/immunogens of this invention. In general, ahost mammalian cell can be any cell that has been efficiently cloned incell culture. However, it is apparent to those skilled in the art thatmammalian expression options can be extended to include organ cultureand transgenic animals. Host mammalian cells useful for the purpose ofconstructing a recombinant mammalian expression system include, but arenot limited to, Vero cells, NIH3T3, GH3, COS, murine C127 or mouse Lcells. Mammalian expression vectors can be based on virus vectors,plasmid vectors which may have SV40, BPV or other viral replicons, orvectors without a replicon for animal cells. Detailed discussions onmammalian expression vectors can be found in the treatises of Glover,DNA Cloning. A Practical Approach, Vols. 1-4 (Oxford, IRL Press, 1985).

Fusion proteins may possess additional and desirable structuralmodifications not shared with the same organically synthesized peptide,such as adenylation, carboxylation, glycosylation, hydroxylation,methylation, phosphorylation or myristylation. These added features maybe chosen or preferred as the case may be, by the appropriate choice ofrecombinant expression system. On the other hand, fusion proteins mayhave its sequence extended by the principles and practice of organicsynthesis.

Vaccines and Immunogenic Compositions

When used in vaccine or immunogenic compositions, the proteins orpeptides of the present invention may be used as “subunit” vaccines orimmunogens. Such vaccines or immunogens offer significant advantagesover traditional vaccines in terms of safety and cost of production;however, subunit vaccines are often less immunogenic than whole-virusvaccines, and it is possible that adjuvants with significantimmunostimulatory capabilities may be required in order to reach theirfull potential.

Currently, adjuvants approved for human use in the United States includealuminum salts (alum). These adjuvants have been useful for somevaccines including hepatitis B, diphtheria, polio, rabies, andinfluenza. Other useful adjuvants include Complete Freund's Adjuvant(CFA), Incomplete Freund's Adjuvant (IFA), Muramyl dipeptide (MDP) (seeEllouz et al., 1974), synthetic analogues of MDP (reviewed in Chedid etal., 1978),N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-[1,2-dipalmitoyl-s-glycero-3-(hydroxyphosphoryloxy)]ethylamide(MTP-PE) and compositions containing a metabolizable oil and anemulsifying agent, wherein the oil and emulsifying agent are present inthe form of an oil-in-water emulsion having oil droplets substantiallyall of which are less than one micron in diameter (see EP 0399843).

The formulation of a vaccine or immunogenic compositions of theinvention will employ an effective amount of the protein or peptideantigen. That is, there will be included an amount of antigen which, incombination with the adjuvant, will cause the subject to produce aspecific and sufficient immunological response so as to impartprotection to the subject from subsequent exposure to an HIV virus. Whenused as an immunogenic composition, the formulation will contain anamount of antigen which, in combination with the adjuvant, will causethe subject to produce specific antibodies which may be used fordiagnostic or therapeutic purposes.

The vaccine compositions of the invention may be useful for theprevention or therapy of HIV-1 infection. While all animals that can beafflicted with HIV-1 can be treated in this manner, the invention, ofcourse, is particularly directed to the preventive and therapeutic useof the vaccines of the invention in man. Often, more than oneadministration may be required to bring about the desired prophylacticor therapeutic effect; the exact protocol (dosage and frequency) can beestablished by standard clinical procedures.

The vaccine compositions are administered in any conventional mannerwhich will introduce the vaccine into the animal, usually by injection.For oral administration the vaccine composition can be administered in aform similar to those used for the oral administration of otherproteinaceous materials. As discussed above, the precise amounts andformulations for use in either prevention or therapy can vary dependingon the circumstances of the inherent purity and activity of the antigen,any additional ingredients or carriers, the method of administration andthe like.

By way of non-limiting illustration, the vaccine dosages administeredwill typically be, with respect to the gp120 antigen, a minimum of about0.1 mg/dose, more typically a minimum of about 1 mg/dose, and often aminimum of about 10 mg/dose. The maximum dosages are typically not ascritical. Usually, however, the dosage will be no more than 500 mg/dose,often no more than 250 mg/dose. These dosages can be suspended in anyappropriate pharmaceutical vehicle or carrier in sufficient volume tocarry the dosage. Generally, the final volume, including carriers,adjuvants, and the like, typically will be at least 0.1 ml, moretypically at least about 0.2 ml. The upper limit is governed by thepracticality of the amount to be administered, generally no more thanabout 0.5 ml to about 1.0 ml.

Peptides of the invention corresponding to domains of the envelopeprotein such as V3 may be constructed or formulated into compounds orcompositions comprising multimers of the same domain or multimers ofdifferent domains. For instance, peptides corresponding to the V3 domainmay be circularized by oxidation of the cysteine residues to formmultimers containing 1, 2, 3, 4 or more individual peptide epitopes. Thecircularized form may be obtained by oxidizing the cysteine residues toform disulfide bonds by standard oxidation procedures such as airoxidization.

Synthesized peptides of the invention may also be circularized in orderto mimic the geometry of those portions as they occur in the envelopeprotein. Circularization may be facilitated by disulfide bridges betweenexisting cysteine residues. Cysteine residues may also be included inpositions on the peptide which flank the portions of the peptide whichare derived from the envelope protein. Alternatively, cysteine residueswithin the portion of a peptide derived from the envelope protein may bedeleted and/or conservatively substituted to eliminate the formation ofdisulfide bridges involving such residues. Other means of circularizingpeptides are also well known. The peptides may be circularized by meansof covalent bonds, such as amide bonds, between amino acid residues ofthe peptide such as those at or near the amino and carboxy termini (seeU.S. Pat. No. 4,683,136).

In an alternative format, vaccine or immunogenic compositions may beprepared as vaccine vectors which express the HIV protein or peptide ofthe invention in the host animal. Any available vaccine vector may beused, including live Venezuelan Equine Encephalitis virus (see U.S. Pat.No. 5,643,576), poliovirus (see U.S. Pat. No. 5,639,649), pox virus (seeU.S. Pat. No. 5,770,211) and vaccina virus (see U.S. Pat. Nos. 4,603,112and 5,762,938). Alternatively, naked nucleic acid encoding a protein orpeptide of the invention may be administered directly to effectexpression of the antigen (see U.S. Pat. No. 5,739,118).

Diagnostic Reagents

The HIV protein or peptide compositions of the present invention may beused as diagnostic reagents in immunoassays to detect anti-HIVantibodies, particularly anti-gp120 antibodies. Many HIV immunoassayformats are available. Thus, the following discussion is onlyillustrative, not inclusive. See generally, however, U.S. Pat. Nos.4,743,678; 4,661,445; and 4,753,873 and EP 0161150 and EP 0216191.

Immunoassay protocols may be based, for example, upon composition,direct reaction, or sandwich-type assays. Protocols may also, forexample, be heterogeneous and use solid supports, or may be homogeneousand involve immune reactions in solution. Most assays involved the useof labeled antibody or polypeptide. The labels may be, for example,fluorescent, chemiluminescent, radioactive, or dye molecules. Assayswhich amplify the signals from the probe are also known, examples ofsuch assays are those which utilize biotin and avidin, andenzyme-labeled and mediated immunoassays, such as ELISA assays.

Typically, an immunoassay for anti-HIV antibody will involve selectingand preparing the test sample, such as a biological sample, and thenincubating it with an HIV protein or peptide composition of the presentinvention under conditions that allow antigen-antibody complexes toform. Such conditions are well known in the art. In a heterogeneousformat, the protein or peptide is bound to a solid support to facilitateseparation of the sample from the polypeptide after incubation. Examplesof solid supports that can be used are nitrocellulose, in membrane ormicrotiter well form, polyvinylchloride, in sheets or microtiter wells,polystyrene latex, in beads or microtiter plates, polyvinlyidinefluoride, diazotized paper, nylon membranes, activated beads, andProtein A beads. Most preferably, Dynatech, Immulon® microtiter platesor 0.25 inch polystyrene beads are used in the heterogeneous format. Thesolid support is typically washed after separating it from the testsample.

In homogeneous format, on the other hand, the test sample is incubatedwith the HIV protein or peptide in solution, under conditions that willprecipitate any antigen-antibody complexes that are formed, as is knownin the art. The precipitated complexes are then separated from the testsample, for example, by centrifugation. The complexes formed comprisinganti-HIV antibody are then detected by any number of techniques.Depending on the format, the complexes can be detected with labeledanti-xenogenic Ig or, if a competitive format is used, by measuring theamount of bound, labeled competing antibody. These and other formats arewell known in the art.

Diagnostic probes useful in such assays of the invention includeantibodies to the HIV-1 envelope protein. The antibodies to may beeither monoclonal or polyclonal, produced using standard techniques wellknown in the art (See Harlow & Lane, Antibodies. A Laboratory Manual,(Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 1988). Theycan be used to detect HIV-1 envelope protein by specifically binding tothe protein and subsequent detection of the antibody-protein complex byELISA, Western blot or the like. The HIV-1 envelope protein used toelicit these antibodies can be any of the variants discussed above.Antibodies are also produced from peptide sequences of HIV-1 envelopeproteins using standard techniques in the art (Harlow & Lane, supra).Fragments of the monoclonals or the polyclonal antisera which containthe immunologically significant portion can also be prepared.

The following working examples specifically point out preferredembodiments of the present invention, and are not to be construed aslimiting in any way the remainder of the disclosure. Other genericconfigurations will be apparent to one skilled in the art. Allreferences, including U.S. or foreign patents, referred to in thisapplication are herein incorporated by reference in their entirety.

EXAMPLES

The following methods were used in the Examples:

Reference Serum Donor Envelope Gene Cloning

The donor of the HIV-1 Neutralizing Serum (2) (Reference 2), availablein the NIH AIDS Research and Reference Reagent Program (Catalog Number:1983) is a participant in a long term cohort study at the ClinicalCenter, NIH (Vujcic et al., 1995). The blood used to prepare Reference 2had been collected in the Spring of 1989. Peripheral blood mononuclearcells that had been cryopreserved from donations obtained approximatelysix months and one year prior to the time of Reference 2 collectionswere used as sources of DNA for env gene cloning. The cells had not beenstored to maintain viability. DNA was extracted using phenol/chloroformfrom approximately 1-3×10⁶ cells from each donation (Quinnan et al.,1998). The DNA was used as template in a nested polymerase chainreaction, similar to that described previously, except rTth was used asthe DNA polymerase, following the manufacturer's instructions (Barnes,1992; Cariello et al., 1991). The DNA was cloned into the expressionvector pSV7d, as previously described (Quinnan et al., 1998; Stuve etal., 1987).

Other env Gene Clones and Virus Pools

The following HIV-1 env clones in the expression vector pSV3 wereobtained from the AIDS Research and Reference Reagent Program,93MW965.26 (clade C), 92RWO20.5 (clade A), 93TH966.8 (clade E),92UG975.10 (clade G) (Gao et al., 1994). The production of env clonesfrom the molecular virus clones NL43, AD8, and SF162 has been previouslydescribed (Quinnan et al., 1998; Adachi et al., 1986; Theodore et al.,1996; Englund et al., 1995). env gene of the Z2Z6 strain was clonedsimilarly, using molecular virus clone plasmid as template in polymerasechain reaction, and cloning the genes into the plasmid pSV7d (Seth etal., 1993). The production of primary isolate env clones fromparticipants in the Multicenter AIDS Cohort Study, designated here P9and P10, has also been previously described (Quinnan et al., 1998). P9and P10-virus pools were prepared by single subpassages of the cellculture media from primary cultures in PHA blasts (Quinnan et al.,1998). The use of molecular virus clones for preparation of virus poolsof NL43 in H9 cells, and NL(SF162) and AD8, in PHA blasts, has also beenpreviously described (Quinnan et al., 1998).

Cell Cultures

The H9 cell line was obtained from Robert Gallo (Mann et al., 1989). TheMolt 3 cell line was obtained from the American Type Culture Collection,Rockville, Md. (ATCC). (Daniel et al., 1988) The HOS cell linesexpressing CD4 and various coreceptors for HIV-1 were obtained from theNIH AIDS Research and Reference Reagent Program, as was the PM1 cellline (Deng et al., 1996; Landau et al., 1992; Lusso et al., 1995). The293T cell line was obtained from the ATCC, with permission from theRockefeller Institute (Liou et al., 1994). The H9, Molt3 and PM1 cellcultures were maintained in RPMI-1640 medium supplemented with 10% fetalbovine serum and antibiotics (Gibco). The HOS and 293T cells weremaintained in Dulbecco's Minimal Essential Medium (Gibco), with similarsupplements, except that the HOS cell medium was supplemented withpuromycin for maintenance of plasmid stability. Cryopreserved humanperipheral blood lymphocytes were stimulated with PHA and used for virusinfections (Quinnan et al., 1998; Mascola et al., 1994).

Reverse Transcriptase Assay

Reverse transcriptase activity was assayed as previously described (Parket al., 1998).

Virus Neutralization Assays

The virus NL43 was used in neutralization assays which employed Molt3cells as target cells and used giant cell formation for endpointdetermination, as previously described (Vujcic et al., 1995). Theamounts of virus used were sufficient to result in the formation of30-50 giant cells per well (Vujcic et al., 1995; Lennette, 1964). Theviruses, NL(SF162) and AD8, P9 and P10 were tested for neutralization inPHA stimulated human lymphoblasts in the presence of IL-2 (Quinnan etal., 1998; Mascola et al., 1994). In the latter assays ten percent ofthe cell suspension was removed each week, fifty percent of the mediumwas changed each week, and medium was sampled twice weekly from eachwell for reverse transcriptase assay. The reverse transcriptase assayswere performed on the test samples from the first sampling date at whichthe non-neutralized control wells had reverse transcriptase activityabout 10-20×background, generally on day 14 or 17 of the assay. Theneutralization endpoint was considered to be the highest dilution ofserum at which reverse transcriptase activity was reduced at least fiftypercent. The Reference Neutralizing Sera 1 and 2 and the NegativeReference Serum were used as positive and negative controls (NIH AIDSResearch and Reference Reagent Program)

Pseudovirus Construction and Assays of Pseudoviruses for Infectivity andNeutralization

Pseudoviruses were constructed and assayed using methods similar tothose described previously (Quinnan et al., 1998; Deng et al., 1996;Park et al., 1998). pSV7d-env plasmid DNA and pNL43.luc+.E-R- werecotransfected into 70 to 80% confluent 293T cell cultures using thecalcium phosphate/Hepes buffer technique, following manufacturer'sinstructions (Promega, Madison, Wis.), in 24 well plastic tissue culturetrays or 25 cm² flasks (Quinnan et al., 1998; Deng et al., 1996; Park etal., 1998). After 24 hours the medium was replaced with mediumcontaining one mM sodium butyrate (Quinnan et al., 1998; Park et al.,1998). Two days after transfection medium was harvested, passed througha 45 μm sterile filter (Millipore Corp, Bedford, Mass.), supplementedwith an additional 20% fetal bovine serum and stored at −80° C.

Pseudovirus infectivity was assayed in PM1 or HOS-CD4 cells expressingvarious co-receptors. Transfection supernatants were serially dilutedand inoculated into cells in 96 well plates, 50 μl per well. Assays wereroutinely performed in triplicate. The cultures were incubated for fourdays, centrifuged at 400×g for ten minutes if PM1 cells were used, andmedium removed by aspiration. The cells were washed twice with phosphatebuffered saline, lysed with 25 μl cell culture lysing reagent accordingto the manufacturer's instructions (Promega, Madison, Wis.); the cellswere then tritated into the medium, and 10 μl of the suspensions weretransferred to wells of 96 well luminometer plates. Substrate was addedin 100 μl volumes automatically, and the luminescence read using aMicroLumatPlus luminometer (EG&G Berthold, Hercules, Calif.). Mock PVcontrols were used in each assay consisting of media harvested from 293Tcell cultures transfected with pSV7d (without an env insert) andpNL43.Luc.E-R-, and processed in the same way as cultures for PVpreparation. Infectivity endpoints were determined by a modified ReedMunch method; an individual well was considered positive if theluminescence was at least 10-fold greater than the mock control, and theendpoint was considered to be the highest dilution at which thecalculated frequency of positivity was ≧50% (Quinnan et al., 1998; Parket al., 1998; Lennette, 1964). Luminescence resulting from infectionwith minimally diluted samples was generally about 10,000-fold greaterthan background.

Neutralization tests were performed using PM1 or HOS-CD4 cells. Aliquotsof 25 μl of two-fold serial serum dilutions were mixed with equalvolumes of diluted PV in wells of 96 well plates. The PV dilutions wereselected so as to expect luminescence in the presence ofnon-neutralizing serum of about 100-fold of background. Assays wereperformed in triplicate. The virus serum mixtures were incubated forsixty minutes at 40° C., after which 150 μl aliquots of PM1 cellsuspensions were added, which each contained 1.5×10⁴ cells, or thesuspensions were transferred to wells containing HOS-CD4 cells. Theassays were then processed similarly to the infectivity assays. Theneutralization endpoints were calculated by a modified Reed-Munch methodin which the endpoint was considered to be the highest serum dilutioncalculated to have a frequency of ≧50% for reducing luminescence by ≧90%compared to the non-neutralized control. PV titrations were conducted induplicate in parallel with each neutralization assay.

Nucleic Acid Sequencing

Nucleotide sequence analysis was performed using the di-deoxy cyclesequencing technique and AmpliTaq FS DNA polymerase, according tomanufacturer's directions (Perkin Elmer Applied Biosystems, Foster City,Calif.). After the sequencing reaction the DNA was purified usingCentriflex Gel Filtration Cartridges (Advanced Genetic Technologies,Gaithersberg, Md.). Sequencing gels were run and analyzed using anApplied Biosystems Prism, Model 377 DNA Sequencer. Sequencing wasperformed on both strands. Sequence alignment was performed using theEditseq SEQMAN, and Megalign programs in DNA Star according to themethod of Higgins and Sharp (1989).

Example 1 Comparability of Clones Isolated from Different Time Points

From the samples of patient cells from each of the two time points, envclones were recovered which encoded proteins which were capable ofmediating pseudovirus entry into target cells. Two such clones from eachtime point were further characterized. As shown in Table 1, theenvelopes of all four clones mediated infection for PM1 cells and wereneutralized comparably by References 1 and 2. Pseudoviruses carryingenvelopes corresponding to each clone were also tested for infectivityfor HOS-CD4 cells expressing either CXCR4 or CCR5, and all four wereinfectious only for the cells expressing CCR5, as shown in Table 2.Nucleotide sequences including the V3 regions were analyzed for eachclone, with more than 300 bases assigned for each, and no differencesbetween the clones were found (results not shown). Based on the absenceof demonstration of differences in these assays, a single clone from theMarch sample was selected for use in subsequent assays, and isdesignated R2, hereafter.

Example 2 Clone R2 Genotype and Host Range Phenotype

The complete nucleotide sequence of the env gene clone R2 was determinedand found to have an open reading frame of 2598 bases (Genbank AccessionNumber: AF 128126) (SEQ ID NO: 24). The amino acid sequence deduced fromthis sequence is shown in Table 3 (SEQ ID NO: 1). There are thirtypredicted glycosylation sites, compared to twenty-nine in the consensuslade B sequence; four consensus glycosylation sites are lacking in R2,including those at residues 146, 215, 270 and 368 (numbering accordingto the Human Retroviruses and AIDS Database lade B consensus sequence),in the V2, C2, C2 and V4 regions of gp120, respectively (Myers et al.,1993). The consensus glycosylation sequences at residues 215 and 270 arehighly and moderately variable, respectively.

Genotypic analyses conducted included evaluation of the gp120 and gp41nucleotide coding sequences in comparison to those of a number ofstrains of clades A through G, as shown in FIG. 1 (Saitou et al., 1987;Myers & Miller, 1988). Both coding regions were more closely related tolade B than non-clade B sequences. Comparative analyses of regions ofthe predicted gp120 and gp41 amino acid sequences were also performed(results not shown). The regions analyzed included: each constant andvariable region of gp120; the proximal gp41 ectodomain including theleucine zipper region; the part of gp41 extending from the end of theleucine zipper to the second cysteine; the remaining gp41 ectodomain,and the transmembrane region; and the cytoplasmic region. R2consistently related more closely with the clade B sequences than theothers.

Example 3 Comparative Sensitivity of R2 and Other Clade B Viruses andPseudoviruses to Neutralization by Sera from Individuals with Clade BInfections

The neutralization of R2 pseudovirus was compared to other lade Bviruses and pseudoviruses as shown in FIG. 2. Of the fivevirus-pseudovirus comparisons made (P9, PI O, NL43, AD8 and SF162 V andPV), there were no significant differences in the neutralization ofmatched viruses and pseudoviruses by paired t test (statistical resultsnot shown). Each of the pseudovirus preparations was neutralized byseven, eight, or nine of the sera tested, and the geometric mean titersranged from 1:13.9 to 1:56, while the R2-PV was neutralized by all tenof the sera tested, with a geometric mean titer of 1:73.5. Although theneutralization titers of each of the different sera against R2 and theother pseudoviruses were frequently within four-fold, the neutralizationof R2-PV was significantly greater by paired t test than four of theother PV preparations.

Example 4 Comparative Neutralization of Pseudoviruses Expressing R2 andOther Envelopes of Diverse Subtypes by Sera from Diverse SubtypeInfections

The results of comparative neutralization testing using sera fromindividuals infected with HIV-1 strains of subtypes A, C and E, and theReference 1 and 2, and one Thai clade B serum are shown in Table 4.Reference 2 neutralized the pseudovirus expressing the homologous R2envelope at the modest titer of 1:64 in the experiment shown and withintwo-fold of this titer in many other experiments. It neutralized theother seven pseudoviruses tested at low to moderate titers, as well. TheR2 pseudovirus was neutralized by seventeen of twenty-four sera,including sera from people infected with each of the clades A-F. Theother two lade B pseudoviruses were neutralized less frequently and werealso neutralized infrequently by the clade E sera. The frequency ofneutralization by sera from individuals infected with different cladeswas not significantly skewed for any of the other four pseudoviruses.Clade A, C, D and G pseudoviruses were neutralized by eight, seventeen,six and three of the seventeen sera tested, respectively. The lade Cpseudovirus was substantially more sensitive to neutralization, ingeneral than the others tested. The dade E pseudovirus was neutralizedby five of five clade D sera and seven of eight lade E sera but only oneof the sera from people infected by other clades.

Example 5 Synthetic Peptides Generated from V3 Amino Acid Sequences fromR2 Strain

R2 strain V3 peptides were synthesized using an automated ABIsynthesizer and FMOC chemistry (Zeng et al., 1997). The sequences ofthese peptides were KSIPMGPGRAFYTTGQI (SEQ ID NO:2) andCSRPNNNTRKSIPMGPGRAFYTTGQIIGDIRQAHC (SEQ ID NO:3). The mutantR2(313-4PM/HI, 325Q/D) V3 peptide was prepared similarly. Strain93TH966.8 V3 peptide, sequence: CTRPSNNTRTSTTIGPGQVFYRTGDITGNIRKAYC (SEQID NO:4) was synthesized using the same methods. The peptides werepurified using C 18, acetonitrile-in-water gradient chromatography witha Waters High Performance Liquid Chromatograph. Sequences of thepurified peptides were verified using an ABI automated sequencer.Peptides were lyophilized and stored at 4-8° C. Preparation of a linearMN strain V3 peptide has been described previously (Carrow et al.,1991). Cyclic MN strain 35-mer peptide was obtained from the AIDSResearch and Reference Reagent Program (Catalog #1841) provided byCatasti et al., (1996).

The R2 V3 35-mer was insoluble in water, while all other peptides testedwere soluble in water to at least 10 mg/ml. To obtain cyclic peptides,solutions of the R2 and R2(313-4PM/HI, 325Q/D) V3 35-mers indimethylsulfoxide (DMSO), 10 mg/ml, were diluted 1:10 in water at roomtemperature or 37° C. and the pH was adjusted to 8.5 with ammoniumhydroxide. These solutions were aerated by bubbling air through thesolutions for periods ≧1 hour. Following aeration, the pH was adjustedto 7.4 using hydrochloric acid. A portion of the R2 35-mer peptideprecipitated during these procedures. To obtain an approximatequantitation of the amount of R2 V3 35-mer that remained in solution,the turbidity of the suspension was determined at 480 nm wavelengthvisible light using a spectrophotometer. The spectrophotometer wasblanked with a solution of 10 percent DMSO in water, and a standardcurve was produced using slurries of known amounts of the 35-mer peptidesuspended in water. The amount of precipitate estimated by turbidity wassubtracted from the amount of peptide added at the beginning of thepreparation procedure to estimate the amount remaining in solution. Thesolubility of the oxidized R2 35-mer peptide in 10 percent DMSO solutionat pH=7.4 was estimated to be 300-350 μg/ml when processed at roomtemperature, or 850-900 μg/ml when processed at 37° C. Peptides weresterilized by passage through 0.22μ pore size filters prior to use.

Example 6 Peptide Blocking of Neutralizing Antibody Activity AgainstClone R2 Pseudovirus

The neutralization blocking effects of synthetic V3 peptides wereexamined to test the contribution of V3-anti-V3 interactions in theneutralizing cross reactivities of Reference 2 and clone R2. Theblocking effects of peptides on neutralizing activity of Reference 2against clone R2 pseudovirus are shown in FIG. 3A. Usually, the linear17-mer peptide had no inhibitory effect on neutralization, as shown. Inonly one of several experiments two-fold reduction of neutralization wasobserved in the presence of 17-mer peptide. Concentration-dependentinhibitory effects of the cyclic 35-mer R2 V3 peptide on neutralizationof clone R2 pseudovirus by Reference 2 was observed in the experimentshown and in numerous other similar experiments. Maximum effect wasobserved at approximately 15 μg/ml. No inhibitory effect was observedusing a cyclic peptide homologous to the V3 region of the HIV-193TH966.8 strain.

The comparative effects of the R2 and MN strain V3 peptides onneutralization of the R2 and MN strain pseudoviruses are shown in FIG.3B. The results shown are representative of two additional experiments.Only the cyclic R2 V3 peptide produced consistent blocking of R2pseudovirus neutralization. The linear R2 and MN, and the cyclic MNpeptides did not block R2 neutralization in two experiments and blockedonly two-fold in a third experiment. In contrast, the MN cyclic andlinear peptides consistently inhibited MN strain neutralization eight-to sixteen-fold in these experiments, and the R2 peptides had consistenttwo-fold inhibitory effects on neutralization of the MN strain. Theseeffects of MN peptides on MN strain neutralization are consistent withprevious reports (Carrow et al., 1991; Park et al., 1999).

Example 7 Cyclic R2 V3 Peptide Inhibition of Neutralization of R2Pseudoviruses by Sera from MACS Patients

Inhibition of heterologous serum neutralization of R2 pseudovirus bycyclic R2 V3 peptide was evaluated to determine if cross reactivity ofthese sera with R2 included effects of anti-V3 antibodies. Thecomparative neutralization titers of sera from ten patients from theMACS against clone R2 pseudovirus in the presence and absence of cyclicR2 V3 peptide are shown in FIG. 4A (Quinnan et al., 1998). These serahave been described previously, and have been shown to neutralizeprimary HIV-1 enveloped pseudoviruses cross reactively, but to a lesserextent than Reference 2 (Zhang et al., 1999). Each serum was testedtwice. Seven of the sera appeared to be inhibited at least two-fold inone or both experiments. The geometric mean inhibitory effect of all thetests was 1.9-fold. The results of twelve tests conducted at the sametimes as those tests shown in FIGS. 4A and 4B are shown for Reference 2;the geometric mean inhibitory effect was 3.56.

Example 8 Cyclic R2 V3 Peptide Inhibition of Reference 2 Neutralizationof Pseudoviruses Expressing Envelopes from the MACS Patients

Inhibition of Reference 2 neutralization of pseudoviruses expressingheterologous envelopes by cyclic R2 V3 peptide was evaluated todetermine whether anti-V3 antibody contributed to the neutralizing crossreactivity of Reference 2. The results of these experiments are shown inFIG. 4B. Each pseudovirus was tested two or three times. The peptideappeared to exert a two-fold inhibitory effect in one, two, or three ofthe experiments using each of the six pseudoviruses. The geometric meaninhibitory effect was 1.6-fold.

Example 9 Induction of Cross-Reactive Neutralizing Antibodies in MiceFollowing Immunization with Recombinant Delivery Vectors Encoding Hiv-1Envelope Proteins

The DNA clone encoding the R2 envelope was introduced into an expressionvector which can be used to express the envelope protein complex in vivofor immunization. The recombinant delivery vector expressing the R2envelope clone was been administered to mice, both in its full length,encoding both gp120 and gp41, or in a truncated form. The truncated formis secreted by cells which express gp140. Both the full-length andtruncated form of these constructs induced neutralizing antibodies inmice. The mice which received the gp140 construct, which includes the V3region, have developed neutralizing antibodies which neutralize at leastthree different strains of HIV-1, including the R2 strain, a macrophagetropic laboratory strain known as SF162, and a primary strain which isnot laboratory adapted. The amount of cross-reactivity observed exceedsthat induced by most or all other HIV immunogens that have been testedas single agents. TABLE 1 Comparative Neutralization of PseudovirusesExpressing Multiple Envelope Clones From Donor 2 Neutralization TiterAgainst Clone Serum 10.1 10.2 3.1 3.2 Reference 1 1:32  1:64  1:32 1:64  Reference 2 1:128 1:128 1:128 1:128

TABLE 2 Coreceptor Dependency of R2 Pseudovirus Entry Into HOS-CD4 CellsInfectivity Titer In HOS-CD4 Cells Expressing Pseudovirus CCR1 CCR2bCCR3 CCR4 CCR5 CXCR4 In PM1 Cells R2 <1:4 <1:4 <1:4 <1:4 1:64  <1:4 1:32P9 <1:4 <1:4 <1:4 <1:4 1:256 <1:4 1:8  NL4-3 <1:4 <1:4 <1:4 <1:4 1:32  >1:256 1:8  AD8 <1:4 <1:4 <1:4 <1:4 1:256 <1:4 1:32

TABLE 3 Inferred Amino Acid Sequence of the R2 Envelope Clone from Donor2. Residue Amino Acid Residue^(a) Number MRVKGIRRNY QHWWGWGTMLLGLLMICSAT EKLWVTVYYG VPVWKEATTT  50 LFCASDAKAY DTEAHNVWAT HACVPTDPNPQEVELVNVTE NFNMWKNNMV 100 EQMHEDIISL WDQSLKPCVK LTPLCVTLNCTDLRNTTNTN NSTDNNNSNS 150 EGTIKGGEMK NCSFNAITSI GDKNQKEYAL LYKLDIEPIDNDNTSYRLIS 200 CNTSVITQAC PKISFEPIPI HYCAPAGEAT LKCNDKKFSG KGSCKNVSTV250 QCTHGIRPVV STQLLLNGSL AEEEVVIRSE NFTNNAKTII VQLREPVKIN 300CSRPNNNTRK SIPMGPGRAF YTTGQIIGDI RQAHCNISKT NWTNALKQVV 350 EKLGEQFNKTKIVFTNSSGG DPEIVTHSFN CAGEFFYCNT TQLFDSIWNS 400 ENGTWNITRG LNNTGRNDTITLPCRIKQII NRWQEVGKAM YAPPIKGNIS 450 CSSNITGLLL TRDGGKDDNS RDGNETFRPGGGDMRDNWRS ELYKYKVVKI 500 EPLGVAPTKA KRRVVQREER AVGLGAMFIG FLGAAGSTMGAASVTLTVQA 550 RQLLSGIVQQ QSNLLRAIEA QQHLLQLTVW GIKQLQARIL AVERYLKDQQ600 LLGIWGCSGK LICTTTVPWN ASWSKNKTLE AIWNNMTWMQ WDKEIDNYTS 650LIYSLIEESQ IQQEKNEQEL LELDKWANKW NWFDISNWLW YIKIFIMIVG 700 GLVGLRIVFVVLSIVNRVRQ GYSPLSFQTR LPAPRGPDRP EEIEEEGGDR 750 DRDRSGLLVD GFLTLIWVDLRSLCLFSYHR LRDLLLIVTR IVELLGRRGW 800 EILKYWWNLL QYWSQELKNS AVSLFNATAIAVAEGTDRVI EVLQRVGRAL 850 LHIPTRIRQG LERALL 866^(a)Amino Acid residues are identified by standard single letterdesignations. Predicted N-linked glycosylation sites are indicated byshading and bolding.

TABLE 4 Neutralization of Pseudoviruses Expressing Envelopes of VariousClades by Sera from People Infected with Various Clades of HIV-1 NATiter Against Pseudovirus (Clade)^(a) P9 P10 RW020 MW965 Z2Z6 TH966UG975 Clade Serum^(b) R2 (B) (B) (B) (A) (C) (D) (E) (G) B Ref 1 32 1632 <10 256 10 <8 <10 Ref 2 64 32 64 10 128 40 8 10 WR8465 20  NT^(c) 80<10 640 10 <10 10 A 37570 320 160 20 80 2560 <10 <10 <10 35374 40 <10<10 <10 640 <10 <10 <10 35837 40 20 <10 80 2560 <10 <10 <10 C  5107 4010 <10 10 1280 <10 <10 <10  5708 10 <10 <10 <10 320 <10 <10 <10  5218 80<10 <10 <10 1280 <10 <10 <10 D UG9240 <10 NT NT NT NT NT 20 NT UG9370<10 NT NT NT NT NT 10 NT UG9386 <10 NT NT NT NT NT 10 NT UG93097 10 NTNT NT NT NT 10 NT UG94118 10 NT NT NT NT NT 20 NT E WR5659 10 <10 <10<10 20 <10 40 <10 WR5901 <10 <10 <10 40 320 10 40 10 WR8177 <10 <10 <1040 640 10 80 <10 WR8657 <10 <10 10 10 640 <10 80 <10 WR8593 <10 <10 <10<10 160 10 40 <10  1008 <10 <10 <10 <10 10 <10 <10 <10  1053 20 <10 <10<10 40 <10 20 <10  1062 20 10 <10 10 320 <10 20 <10 F BR9318 <10 NT NTNT NT NT <10 NT BR93019 10 NT NT NT NT NT <10 NT BR93020 20 NT NT NT NTNT <10 NT BR93029 10 NT NT NT NT NT <10 NT^(a)Neutralization titers are the dilutions at which 90% inhibition ofluminescence was observed.^(b)Sera were the Reference Neutralizing Human Serum 1 and 2, or wereprovided by Dr. J. Mascola, HIVNET, or the UNAIDS Program, as describedin the text.^(c)NT = not tested.

TABLE 5 Comparison of V3 Region Amino Acid Sequences of Clone R2 withPhenetic Subgroup Consensus Sequences 1 Through 13 and Clade A Through EConsensus Sequences.^(a) Clone, Subgroup or Clade V3 Region Amino AcidSequence R2 NNTR.KSIPMGPGRAFYTTGQIIGDIRQAHC PHENETIC 1 (SEQ ID NO: 6)----.---HI----------D---------- PHENETIC 2 (SEQ ID NO: 7)----.---SI-------A--E---------- PHENETIC 3 (SEQ ID NO: 8)----.---SI-------A--K---------- PHENETIC 4 (SEQ ID NO: 9)----.---RI---Q---A--D---------- PHENETIC 5 (SEQ ID NO: 10)----.---HI-------A--K---------- PHENETIC 6 (SEQ ID NO: 11)K--RRR-H.I---------K----------- PHENETIC 7 (SEQ ID NO: 12)----.T--TI---QV--R--K---------- PHENETIC 8 (SEQ ID NO: 13)KKM-.T-ARI----V-HK--K---S-TK-Y- PHENETIC 9 (SEQ ID NO: 14)----.Q-THI---A-L---.D---K------ PHENETIC 10 (SEQ ID NO: 15)----.QGTHI-----Y---.N---------- PHENETIC 11 (SEQ ID NO: 16)----.QRSTI-Q-QAL---.E-R------A- PHENETIC 12 (SEQ ID NO: 17)D-IKIQRT-I-A-A-L---RITGYI.G---- PHENETIC 13 (SEQ ID NO: 18)Q-K-.QGT-I-L-Q-L---R.-K----K--- CLADE A (SEQ ID NO: 19)----.--VHI---Q---A--D---------- CLADE B (SEQ ID NO: 20)----.---HI----------E---------- CLADE C (SEQ ID NO: 21)----.---RI---QT-YA--D---------- CLADE D (SEQ ID NO: 22)----.QRTHI---Q-L---.R---------- CLADE E (SEQ ID NO: 23)----.T--TI---QV--R--D------K-Y-^(a)Dashes indicate residues at which the individual sequences areidentical to R2. The periods indicate sites of insertions or deletions.

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1. An isolated HIV envelope protein or fragment thereof which, whenadministered to a mammal, induces the production of broadlycross-reactive neutralizing anti-serum against multiple strains ofHIV-1.
 2. An isolated HIV envelope protein comprising the amino acidsequence of SEQ ID NO:
 1. 3. An isolated HIV envelope protein orfragment thereof comprising a proline at a position corresponding toamino acid residue 313, a methionine at a position corresponding toamino acid residue 314 and a glutamine at a position corresponding toamino acid residue 325 of SEQ ID NO:1.
 4. An isolated HIV envelopeprotein or fragment thereof comprising a V3 region having the amino acidsequence P M X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀Q, wherein X₁-X₁₀ are anatural or non-natural amino acid.
 5. A vaccine composition comprisingan isolated HIV-1 envelope protein or 20 fragment thereof of any one ofclaim 1 and a pharmaceutically acceptable carrier.
 6. An immunogeniccomposition comprising an isolated HIV-1 envelope protein or fragmentthereof of any one of claim 1 and a pharmaceutically acceptable carrier.7. An isolated nucleic acid molecule encoding the HIV-1 envelope proteinor fragment thereof of any of claim
 1. 8. A fusion protein comprisingall or a portion of a microbiological antigen into which any one of theproteins of claim 1 has been inserted.
 9. A recombinant delivery vectorencoding a fusion protein comprising all or a portion of amicrobiological antigen into which any one of the proteins of claim 1has been inserted.
 10. A vaccine composition comprising any one of therecombinant delivery vectors of claim 9 and a pharmaceuticallyacceptable carrier.
 11. An immunogenic composition comprising any one ofthe recombinant delivery vectors of claim 9 and a pharmaceuticallyacceptable carrier.
 12. A recombinant delivery vector encoding anattenuated virus further comprising a nucleotide sequence encoding oneor more of the proteins of claim
 1. 13. The recombinant delivery vectorof claim 12 wherein the attenuated virus is selected from the groupcomprising HIV, encephalitis virus, poliovirus, poxvirus and vacciniavirus.
 14. A vaccine composition comprising any one of the recombinantdelivery vectors of claim 12 and a pharmaceutically acceptable carrier.15. An immunogenic composition comprising any one of the recombinantdelivery vectors of claim 12 and a pharmaceutically acceptable carrier.16. A method of generating antibodies in a mammal comprisingadministering one or more of the proteins or fragments thereof of claim1, in an amount sufficient to induce the production of the antibodies.17. A method of generating antibodies in a mammal comprisingadministering a DNA or mRNA sequence encoding any one of the proteins orfragments thereof of claim 1, in an amount sufficient to induce theproduction of the antibodies.
 18. The method of claim 17, wherein saidDNA is naked DNA.
 19. A diagnostic reagent comprising one or more of theisolated HIV-1 envelope proteins or fragments thereof of any one ofclaims 1-4
 20. A method of detecting HIV-1 antibodies in a samplecomprising the step of determining whether antibodies in the sample bindto one or more of the HIV-1 envelope proteins or fragments thereof ofclaim
 1. 21. A cyclic peptide comprising the amino acid sequence ofclaim
 3. 22. An isolated antibody which specifically recognizes theprotein of claims claim 3.